The objective of this application is to understand the molecular mechanism by which cytotoxic lymphocytes kill target cells such as virus infected cells and tumor cells. Lymphocyte mediated cytolysis may be of importance for protection against cancer by immune surveillance systems. In addition it is one of the fundamental defences of the immune system against virus infection. Based on the observation that granules isolated from killer lymphocytes are highly cytolytic, that they cause the release of cytoplasmic and nuclear (DNA) markers from attacked targets and that they form two types of membrane lesions on target cells, it is postulated that granules contain the entire or at least a large part of the killing mechanism of cytolytic lymphocytes. It is therefore planned to study the composition of granules and to isolate from granules the molecules responsible for formation of membrane lesions and for DNA degradation in target cells. In addition, the characterization of all granule proteins may result in the discovery of new activities. It is also planned to study the granule membrane and to analyze the regulation of the release of granules by killer lymphocytes in the cytolitic reaction. Based on the finding that killer lymphocytes can lyse several target cells in succession the question will be addressed how cytolytic lymphocytes escape the destructive force of their own cytolytic granules during the cytolytic reaction. Granule proteins will be characterized by conventional biochemical and immunological techniques and this information will be used to obtain cDNA clones from an expression cDNA library of cytolytic lymphocyte clones. Cloned cDNA will be used for structural studies by DNA sequencing, for the expression of encoded proteins in E. coli, and for the preparation of monospecific antisera. DNA probes will also facilitate the analysis of the regulation of cytolytic granule proteins in related studies. These projects should result in a more detailed understanding of the molecules of cytolytic granules responsible for cell killing, which in turn may be useful for immunotherapeutic or diagnostic approaches to cancer, autoimmunity, and transplantation.